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CA 02619245 2008-02-12
WO 2007/019621 PCT/AU2006/001166
CHIMERIC ANTIBODIES WITH NEW WORLD PRIMATE REGIONS
FIELD OF THE INVENTION
The present invention t'clates to a chirneric ant.i.body or anti.gen-binding
portion thereof,
wherein the ant,igen binding portion compriscs at least two eomplementarity
d.eterminirlg
region (CUR) sequences and at least threc framework rcg.ions, whcrein at least
one CDR is
a New World primatc Goft, and to the use of the antibody or antigen-binding
portion
thereof in treating diseases or disorders.
BACKGROUND OF THIc INVENTION
AnLiboclies (immunoglobulii,s) play an important role in the immunc system of
a mammal.
They are produced by plasma cells which have developed from precursor B cells.
Antilxuiies consist of two identica3 light polypeptide chains and two
icientical heavy
polypeplide chains which are joined by tlisul3'ide britlges. The light chains
are referrcd to
as either kappa or lambda light chv.ins and the heavy cllains as gamina, mu,
delta, alpha or
epsilon. Each chain consists of a constant a d variable region. The variable
region gives
the antibody its 5pecificity. Within each variable rcgion ai-e regions of
hypelvari ability or
Comple;iTlentatiLy determining regions (CDRs) which are flankcd by morc
conserved
regions refeiTed to as framework regions. Within eaclt variable region are
three CDRs and
four framcwork regions.
Antibodies are bifunctional t-0oleculcs, the N-tct'minal variable segments
from the heavy
and light chains associate together in a specific manner to generate a three-
dimensional
structure with affinity for a particular epitopc on tha surface of an antigen.
The constant
rcgion. segments are responsible for prolcmged serum half-life and the
effector fnnctiuns of
the a.ntibody and relat.e to ccmplement bindirig, stimulation of phagocytosis,
antibcxiy-
del:tendent. cellular cytotoxicity and triggeritig of grantilocyte granule
release.
The development of hybridotna technology has facilitated the production of
monoclonal
antibodies of a particular speeificity. Typically, such hybridomas are murinc
hybridomas.
Hutnan/mouse chimaric antiboclics have been created in which antibody variable
region
sequences from the mouse genoinc are combincd with antibody constant i-egion
sequences
fram the human genome. 'I'he chitneriG anlibodies exhibit the binding
characteristics of the
parental mouse antibody, and the cffector functions associated with the human
constant
rcgion_ The antibadics a.re produccd by expression in a host ceIl, inoluding
for example
Chinese Hamster Ovai=y (Ct1=O), N5U myeioma cells, COS cells and SP2 cells.
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2
Stich chimeric antibodies have been used in humail theieipy, however
antibodies to these
eliimeric antiboc3ics have been produced by the hui-nan recipient. Such anti-
chitnaric
antibodies are detrimental to continued therapy with chimeric antibodies.
It has heen suggested that human monoclonal antibodies are expected to be an
improvement over mouse rnonoclonal antibodies for in vivo human therapy. From
work
done with antibodies from Old World primates (rhesus monkeys and chimpanzees)
it has
been postulated that these non-ht.nnan primate antibodies will be tolerated in
humans
because they are stiiIcturally similar to human antibodics (l;tu=lich PH eL
al., I-luman and
primatr nionoclonal antibodies for in. vivc) therapy. Clin Cbem. 34:9 pg 1681-
1688 (1988)).
Furthertnore, because human antibodies are non-irr:tnttnogenic in Pdhcsus
xnonkeys (Ehrich
PH el al., Rhesus tnonkey responses to multiple injections of human monoclonal
antibodies. Hybridoma 1987; 6:151-60), it is likely that the converse is also
applicable and
primate antibodies will be non-irxtmunogenic in lzumans. These monoclonal
antibodies are
secreted by hybridomas constructed by htsing lymphcx:ytes to a human x mouse
heteromycloma.
EP 0 605 442 discloses chitneric antibodies which bind hunlan antig ens. These
antibodies
ccsmpriso the whole variable region from an Old World molikey and the constant
region of
a human or chirnpancee antibody. Ona; of the advantages suggested in this
reference for
these constructs is the ability to raise antibodies in Old World monkeys to
human antigcns
which aTe less immunogenic in humans coznpetrecl with antibodies raised in a
mouse host.
Ncw World primates (infranrder- Platyrrhini) comprise at teast 53 spccies
commonly
divided into two families, the Cullith.ricidcze and Ccbidae. The
CalliXFtriciclae consist of
marmosets and tamarins. The Cehictae inc;ludes the sguirrel tnonkey, titi
monkey, spider
monkey, woolly monkey, oapuchin, uakaris, sakis, ni ght or owl n3onkey and the
howler
monkey.
I'.volutieniuily distanl primates, such as Ncw World prirnates, aA=e not only
sufticiently
ditferent from htanans to allow antibodies against human antigens to be
generated, but are
sufficienlfy similar to humans tohave antibodies similar to human antibodies
so that the
host does not generate an anti-antibody imrnune response when such primate-
clcrived
antibodies are intt'oduced into a human.
Previous stuclics have charactcrised the expri ssed immuno-globulin heavy
chain i~cpertoire
of the Callithrix jac.chus marinoset (von Budingen H-C et al.,
Characterization of the
expressed immunoglobulin IGHV repertoire in the New World marmoset Callithrix
CA 02619245 2008-02-12
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3
jacchus. Immunogenetics 2001; 53:557-5G3)_ Six IGHV sul.}groups were
identified which
showed, a high degree c7C= sCClUeilce sinlilat'ity to lhoir human ICl iV
counterparts. '1'he
framework regions wm tl7orc coilserved when compared to the com[iletnentarity
detennining re&ns (CDRs). The degree nf similarity betwcen C. jacchus and
human
IGHV sequences.was less tllait between non-human Old World primates ancl
humanq.
1)amain antibodies
Domain antibodies (dAb) are functit>nat binding units which can he created
using antibody
frEtincworks and correspond to the variable regions of aither the heavy (VH)
or light (VL)
chaiiis of antibodies. Domain antibodies have ainolecular weight of
approximat.cly 13
kDa, or les5 than one lenth the size of a full antilwdy.
lmmune>gluFiulin light chains are referred tc) as either kappa or lambda light
ehains and the
heavy chains as gainma, mu, delta, alpha or epsilon. The variable region gives
the
antibody its specificit.y. Witi,in each variable region are regions of
hyperva.riabilily,
otherwise lciiown as coi.nplementarity deterrnining regions (CDRs) which are
flanked by
more oonscived regions referred to as framework regions. Within each light and
heavy
chain variable region are thi-ee CDRs and four framework regions_
In coutrast to conventional antibcxlies, domain antxbodies are well expressed
in baotciYal,
yeast and srtammalian systems. Their small size allows for higher rnolar
cluarttities per
gram of product, thus providing a sigiiiYicatlt increase in potency. In
addition, domain
antitrodies can be used as a building block to create therapeulie products
such as multiple
targeting domain antibodies in which a constnict containing two or inore
variable dornains
bind to two or morc therapeutic target%, tir domain antibodies targeted for
pulmonary or
oral ac3miniscration.
SUMMARY OF THE INVENTION
The present inventors have found that New World primat.es provide a rich
source of
binding domains for antibodies against a rangc of antigens including ktuman
antigens.
Further, due to the similarity of the sequenoes between liunian and New World
primates it
is likely that these New World primate sequences will have relatively low
immunogenicity
in humans.
In a lirst aspcct thc present invention provides a chirncric antibody or an
antigen-binding
ptfrtion themof, wherein the antigen-binding portion comprises at least two
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4
cornplernentrjrit.y deterriiining regions ((.t7R) and at least three
fritinework rcgions, wherein
at least one CDR is a Ncw World primate CDR.
In another nspect the present irtvctition providcs ainethod of prodttcing a
cliiineric
antibody or an antigcn-binding portion thereof, the method comprising deleting
a CDR
from << human aniibocly variable region comprising at least two CD.Rs and at
least thru;
framework i-cgions and replacing it with ti New World primate C.C.1R predicted
to he of low
iminunogcnicity to produce a chiineric variatxle regiori,
ln a related a5pcct ttie metliod further coinpri5es the step of reccrvering
the chimcric
variable region.
In yet another aspect the present invention provides a chimeric antibody or an
a.ntigen-
binding portion thereof produced according to the mcthod of thc present
inventiOn.
In a furthcr aspect, the inventioi] provitles a pharmziccuticatl composition
comprising an
etTecti vc amount of the antibody or antigen-binding portian thereof according
to the
pre:;ent inventiori, together witli a phat-rnaceutically acceptable excipient
or diluent.
Jn a still t'urther aspect, the inve.ntion provides for the use uf an
antibcxly or antigen-binding
portion t.hei-eUf of the present invention in a diagnostic application for
detecting an antigen
associated with a particular disease or disorder.
In another rtspcct, the present invention provides a method for treating a
disease or disoi-der
characterised by human TNF-a activity in a htiman subjcct, comprising
auiministering to
the subject in need thereof an effective amount of a chimeric antibody as
described herein,
or a pharmaceutical composition thereof in which the antibody or antigen-
bindi=ng lwrtion
t.hereof binds 'i'NF-cx.
BRIEF DESGRIPTlON OF THE FIGURES
Figure 1 de.monstrates the binding of AB138 to rat MOCY present in rat spinal
cord lysate
(lane 2) and not to CHOICI SV lysate (lane 3). Lane 1 contains molecular
weight markers,
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S
1+'igurc 2 demonstrates the lack of non-specific binding of an attti-TNFa
mcmot;IontiI
antibody ttt the stInle sam.plo of rat MOO presenl in rat spinal cord lysate
(lanr: 2) ancl
C.ti(}K1,1W lysate (lane 3). Lane I contains molecular weiglit markers_
Figure 3 shows the acceptor dc>niain antibody ttmino acid and nu.c)eotide
seclucncc (both
strand5), The restriction digest sites for Kpn I tuld Sar1 DI, which excises a
rCt;ion
includin g the CDR2, is indicated in the figurc. CDR2 residues are indicated
in unclcrlinc.
Figut'e 4 is a sequence alignment of thc domain tlntibod.y acceptor sequence
witll a panel
of New World primate derived imtnunoglobulila sequences perfttrmeci using
AlignX
(Vector NT), Invilrogen, Australia). The C;DE22 is highlighted in bold text.
iL1ETA1LFD a'ESCI~t1PT1C1N OF THE 1NVENTION
In a first aspect the l.tresent invention providcs a chitneric t-ntabody or an
c;ntigen-bislclitlg
Iwrlion thereof, wherein the antigen-bintling portion cotrtprises at least two
complementarity detennining regions (CDR) and at least tiu-ee framework
regions, wherein
at least one CDR is a tYew World primate CDR.
It is preferred that the antigcrl binding portion comprises three CDRs and
four fi~unework
regions. It is also preferred that the ant.igetl-b,inding Portion comprises at
least one, .Znd
prefcrably two human CDRs.
Itt some embodiments of the present invention, the chimeric ttnlibody or
ant,iigen-binding
pottion thereof c:omprises one New World primate CDR. In other cmbod.iments,
the
chimeric antiborly or antlgen-binding portion thereoF compiYses two New World
primate
C131'Zs. In other embodiments CDR2 of the antibody or antigen-binding portion
is a New
World priinatc CD.R.
Tn other cmbodinlents of the present invctltion the at least one New World
primate CDR is
not from a sequence that hinc3s a target atltigen.
(n other embodiments of the prescnt invention the framework regions are human
sequences. Framework regions that are httman sequences incltacEe sequences
derived from
hurnan framework regions, or synthetie sequences based on human framework
regions.
It is within the scope of the present invention, that the sec}ttcnce of the
antigen binding
portion may bc futrther subject to affinity maturation in order t.o .unprove
its antigen binding
charac:teristics such as antigen binding or potency.
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G
An increase in binciatlg is clcmonstratcd hy a[lccrease in KD (lcf~E:,,,,) for
the antibody or
antigen binding portion thereof. An increa,5c in potency is tlemonstrtitcd in
biological
assays. For example, assays that can be used to nlea.sure the potency of the
antibody or
rintigen-hincling portion thereof include the TNFa-induccd L929 cytotoxi.city
noutralisation
S assay, EI-12-incluced human PHA-activated peripheral blood mcanuntIclear
cell (PBMC)
proliferation assay, and RANKL mediated osteociast diffcrentiation of rnouse
splenocytes
(St.ern, 4'roc. Natl. Acad. ,5ci. USA 87:5808 - 0812 (1990); Kong, Y-Y, et at,
Nature
397:315 - 323 (1990); Matthews, N. and M.L. Neale in Ly1nplaokines ccrid
fhite.rJi>J'orts, a
Practical Apprnach, 1987,1VI.J. Clemens, A.Cl. Morris and A.J.H. Gearing,
eds., aR L
Press, p. 22.1)
In a further prufcrred embodiment at 1ea.st cyne framework region is modified
to incrcase
binding iind/or to reduce predicted immunogcnicit.y in hutnans.
In another embodimeni at least one t''.UR scquence is rnodifacd to inc:rease
binding or
potency and or to reduce nredicted immunogenicity in hunaans. It is preferred
that where
at least one C:DIt scquence which is modified it is not the New World primatc
CDR.
Where two ormorc New World primatc CDRs are present then it is preferred that
at least
one Ncw World priniate CDR is not modified.
ln uthcr embodiments of thc present invention at least one framework region is
niodiCicd,
in addition to at least one CDR sequcncc, to increasc binding and or to reduce
predicted
immunogenioity in humans. It is prc.fcrred that thc at the le<<st one CDR
secluence which is
modified it is not a New World primate !C:DP, seclu4nce.
In a preferrcd embodimcnt the antigen-bincling pot-tion is a ciomain
arttibody.
In a further embodirncnt of the piesent invention, the domain antibody tnay be
Inultimorised, as for example, hct.ero- or.homodimer5 (c.g., VH/VH, VL,/VI, or
VH/VL),
Iietero- or homotrimers (e.g., Vlr/VtilVH, VL/VIJVT,, VnlVx)Vi, or VH/VI/V,,),
hetero- or
homotetramers (e.g., V11/VHN11/VI1, Vi1VtIVt/Vr., VHfVif/VB,V1., VH/VII/V1.1VL
or
VH/VJVJVI), or higher order hetero- or homomultimc;rs. lvaultimerisation cdn
increase
the strength of antigen binding, wherein the strength of binding i:; related
to the sum of the
rinding affinities, or part thereof, of the multiple binding sites.
Thus, the invention provides a domain antibody wherein the domain antibody is
linked to
at least one further domain antibody. Each domain antibody may bind to the
same or
rlifferent antigens.
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7
The domain antibody multimurs may furthe.r comprise one or mni-e domain
antibodies
wltich are linked and wherein eacb domain antibody hinds to a differcnt
anligen, tnuiti-
spccific ligands including so-called "dual-specific ligands". For exaratple,
the dual specific
ligands may compriso a pair of Vlt domains or a pair af VL domains. Such dual-
specific
ligands aTe dcscribed in WO 2004/003019 (t'CT/GT32003/002804) itt the name of
Domantis Ltd incorporated by referencc herein in its entirei.y.
Preferably, t.he i4ntibody or atlt.igen-binding portion furlhcr coinprises a
human o.r non-
hu.ma.n priinate constant region sequctice. Examples of non-human primates
inciude, but
ai-e not linlited lo, chimpanzees, uranguattings and baboons.
The present invcntion also pi-ovidcs a method of producing a cliimeric
antibody or an
zitlt,igen-bindittg pcxlion thereof, the method camprising deleting a CDR from
a httman
antibody variahle rcgion comprising at least lwo CDIZs atYd at least tluee
frarnework
regions and .rep lacing it with a New World primate CDR pa-edicted to be of
low
iXnmunogeJlicity lo produce a chimeric variable region.
Jn a related aspect the method furlhor compriscs the step of reeovcring the
chitnerie
variablc iegion.
It is preferred that the selected New World primate CDR is CDR2. It is
prcferred that the
CDR2 sequence is selectedfrctm KVSNRAS, RVSNRAS, KVST.RGP, AASN'IZAS,
TSSNLQA, DASSLQP and YASFLQG. Pnrticularly preferrcd sequences are KVSNRAS,
AASNRAS, TSSNLQA and KVSTRCiP rlue to their predicted lower iinmunogenicity.
In further embodiments the method further comprises motlifyi ng thc sequence
of the
ch.imeric variable rogion to increase binding and/or to decrcase
immunogenicity in humans.
It is pmfcired that the New World pritnate CDR sequence is not modified. Where
two or
more New World pritnate CDR sequences are present then it is ptt;ferred that
at least one
New World primate CDR is not modified_
in other embodiments of lhc present invention at least one framework region is
mc7dified in
addition to at least one CDR sequence, to incrcase binding and or to reduce
predicted
immunogenicity in humans. It is prefetred that the at the least one CDR
sequence which is
n1c-dified it is not a New World primate Cl7R scquence.Thc present invention
also provides
a chimeric antibody or an antigen-hinding portion thereof produced by the
rnethod nf the
present invention.
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8
The tcrm "antibody" fts usecl herein, is intended to refer to immunoglobuiin
txzolectllcs
cotnptYsecl of four polypcptide chains, two heavy (H) chains and two light
(i,) clinins inter-
connected by disulfide bonds. Each heavy chain is comprised of a heavy chain
variable
region (1=1CVR or Vtt) ttttd a heavy chain ccrostant region. 'i'he heavy chain
constant region
$ compiises tltrce domaitls, CH1, CH2 and CH3. Each light chain is crniiprised
of a liglZt.
Ghain variable rcgion (LCVR or VL) and a light chain ccanstant negican. '1'he
light chain
constant region is coinprised of one domain, CL. The VH and Vt, regions cttn
be futther
suhdivided into regions of hypt;rvariability, tCrmed complementarity
determining regiotts
(C):7R), interspersed with, reg.ions that. are morc conscrved, termed
frarnework regions (FR).
Each VH and Vi,iwcomposed ofth.t'ee CDRs and four FRs, arrangcd from aminu-
terminus
to carboxy-terminus in the following order: FR.1, (;DR1, FR2, CDR2, FR3, CDR3,
FR4.
The term "antigcn-binding portion" of an antibody, a.5 used herein refet:s to
one or moro
components or derivatives of ati initnur,oglobulin that exhibit the ability to
hind to an
antigen_ It has, 1:ieen sltown that tllc antigcn-binding function of an
antilx~dy can he
perfonned by fragments of a full lengt.h antibody. Examples of binding
fragrnents
encompassed within the tean "antigen-binding portion" of l3 ri antibudy
include (i) a Fab
fragment, a monovalent frag,in.ent consisting af t.hc VL, VH, CLand CHI
clomains; (ii) a
F(ab')2 fragment, a hivalent frag.m~,ent. compiYsitig two Fab fragments linked
by a disulfide
bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1
c3omains; (iv) a
Fv fragment consisting of the Vi, and Vft domains of a single artn of ttri
aritibcxiy; (v) a dAb
fragmcnt (Ward et a1, 1989, Nature 341:S44-546) wliich consists of a single
V,., domain, or
a VL domain (van don Beuken T et al, 2011, J. M.ol.l3iol, 310, 591); and (A)
an isolated
counpletnentarity detertnining region (CDR). Fuithetm--ore, although the two
domains of
t.he F'v feagzxtent, VL and VH, arc coded by separate genes, they can be
joined, using
recornbinant methods, by a synthetic linker that enables them to be made as a
single
protein chain in which the VL and Vi.l rcgions pair to form monovalent
molecules (known
as single chain Fv (scl7v); (see eg Bird el ul., 1988, Science 242:423-426 and
ilust.on ct a.l.,
19$8 Prctc. Nati. Acad. Sci. USA 85:5879-5883). Such single chain Fvs are also
intended
to he encompassed within the term "antigen-binding perrtion . of an anti body,
Other forlns
of single chain Fvs and related molectilcs sttch as diabc)dies or triabodies
are also
encoinpassed. Diabodies arc bivalent antibodies in which VH and VL doniains
arc
exptvssed on a. single polypt;ptidc chain, but using a linker that is too
sliort to allow for
pairiizg between the two doinains on the same uhain, thereby forcing the
domains to pair
with complementary domains of anothcr chain and creating two antigen binding
sites (see
e.g., J.lolligei-, P., Gat al., 1993, Proc. Nat]. I1cad. Sci. USA,
90:(444.6448; Poljak, R.J., et
rx6., l 994, Stiucture, 2:1121-1123).
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As used herein the term claimeric" means that the antibody or antigcn-binding
portion
includes seyueiices 'from. two diffet-ent species.
Tn one emboditnent, the doniain antibody comprises a ht.tman frttmework
regions and at
lett,st uneNow World primate (.,DRs, mot-e pi-eferably marmoset CDRs.
Prefc;rably, the New World primate is selected from ihe group consisting of
martnoGets,
tamariris, squiirel mcrnkey, titi monkey, spider monkey, woolly nlotll.ey,
capuchiti, uakaris,
sakis, night or owl monkey and the howler monkey. More preferably, the New
World
primate is a marmcaset.
Methods Of produeing chimeric antibodies accordirtg to the invention will be
familiar to
I(,1 persons skilled in the art, see for example, US Fatent Nk7. 4,816,567, US
Patent No.
5,585,089 and US 20030039649 which are incorporated herein by ruferettce in
their
ontirety. Such methods require the use of standard recombinant techniques.
It is piefe.cred that the antibody or antigc:n-bincling portion tlicreof
according to the present
invent.ion ha.s predicted low immunobcnicity in a human Itost.
By "low imrrtunctgenicity" it is mettnt thal the antibody does not raise an
antibody response
in at. least the mttjority of individuals recciving the antibody of sufficient
inal;nitude to
reduce the effectivencss of cotttin.ued administration of the antibody for a
sufficient time to
achieve therapeutic cfficacy.
't'he level of immunogenicity in hu.tnans roay pcedicted using the MHC class
II bindi-ng
prectiction program FrQp'ed. (http_//www.imtech.res.in/raghava/propred) using
a 1 !0
threshold value atialysis of alI alieles. Otherprograms which may lx used
iticlude:
Rankpep (httl)://hicy.dfci.harvard.edu/Too1s/rankpep.html)
.FPiha.se (Algonomics prcaprit;tary suftware: algonomics.com)
Low imm.unogenicity molecules will contain na or low ntimbers of peptides
predicted to
bind to M.llC class 11 alleles that are highly expressed in the target
popttlation (Flower DR.
Doyt.chinoya fA. (2004) immunoinfortnatics and the prediclion of
irnrnunogcnicity, Drug
Discov Today, 9(2): 82-90).
Reduecd .it7xniunogenicity molecules will contain nn or a reduced numbers of
peptides
predictcd to bind to MHC class 11 alleles that are highly expressed in l.hc
target popillation,
relativc to t.he starting donor mnlecule_
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I {}
Funct.ional ttrialysis of MHC class tI bitlding can be perforined by
generating overlapping
pCptides correspnn[ling to the protein of interest and testing these for their
ability to evoke
T cc11 activation (T cel l proliferation assay) or displace a reporter
peptide, a ljinwn MI.IC
class !}-bindtng ptptide (Hammer J cr r.td., 1994, J. l;'xp. Med., 1$0:2353).
,ri The pre4ent iDveniion is further basecl on at method fer ampiitioation of
New World
pYYiTlate irritTtutloglobulin genes, for cxample by polynterase chain
reactiott (PCR) from
nncleic acid exlracted from New World primate lymphocytes using primers
specific for
heavy and light chain variable region gene families. The amplifxed variable
region is then
cloned into an expression vector containing a human or primate canstatlt
region gene for
the production of New World primate chitxieric rccoinhinaint at)tibotly.
Standard
recombinant DNA methodologies are ta sed to obtain antibody heavy and light
chain genes,
incorpnrate these genes into recombitiant expression vectors and ititroduce
the vectors into
host cells, szich as those described in Sambrook, hritsch and Maniatis (eds),
Molecular
Cloning: a.fatxiratory m:.3nual, second edition, Cold Spring t-Iarbor, N.Y
(1989).
Suitable c:xpression vectors will be familiar to those skil.led in the art.
The New World
pi-imatc lymphocytes producing the immunogiohulins.,xre typically immrtalised
by fusioYi
with a tnyeloma cell line to generate a hybricloma.
Preferred mammalian host cells for expressing the recon-ihinant antiboclies of
the invention
inclucle Chinese l-lamster Ovary (C'.FCO), NS() mycloi-na cells, COS cells and
SP2 cel Is.
In addition to nnammalian expression systems, the pi=esent invention also
c:ont.einplatcs the
u5C ofnon-nlamtY-alian expression systems such a5 thcrsL which arc plant or
prokaryotic
(l.i;tcteriktl) derived. Such expression systems would be familiar to peracans
skilled in the
art.
The repertoire of VIt, VL and constant region domains can he a tiaturally
occurring
repertoire of irntnunoglobulin seclttence:; or a synthetic repertoire. A
naturally occtu=i-ing
repertoire is one prepared, for exatnpJe, from immunoglolaulin expressing
cells harvested
from one or more primatGs. Such repertoirCs can be i3a7ve ie. prepared from
newborn
immunoglobulin expressing cells, or rearranged ie. prcpat=ed from, for
oxample, adult
primate B cells. If desired, clones icleirtified from a natural repcltoire, or
any repe;rtoire
that hind the target flntigen are then subject to mutag,c.'nesis and further
scrcening in order
to produce and select variattts with improved binding characteristics.
Synthetic repertoires of iinmunoglobulin variable domains are prepared by
artificially
inta'oducing diversity into a cloned variable doniain. Such affinity
maturation techniques
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11
will bo f-amiliar to persons skilled in the art (Irving R.A. et al. (2001)
Ribosome display
and affinity maturation: from antibodies to tiingle 'V-doniains and steps
towaixl.s cancer
t.hcrttpoutics, Journtil of Immunc>lugical Methoda, 248: 31-45).
The variable region, or a CDR thGreof, of a New World primate antiheidy geme
txt.ay be
cloned by providing nucleic acid eg. eDNA, providing a primer ct>mplement.a.ty
to tlte
cDN.A seclucncc cttcodinl; a 5' lcaclcr scqucncc of an antibody gene,
contacting that cl]'NA
and the pritiuer to forni a hybricl complcx and amplifying the cDNA tc} pt-
oduce nucleic acid
enccxiing the variable region (or CDR region) ot thc NCw Worltl primate
antihody gene.
It will be appreciated by persons skilled in the art of the present invention,
the non-New
World primate variable reg,icro sequence may be used as an acceptor for
grafting Ncw
World pritstatc sc;quences, in pitrtiuull1r, CDR sequences using standard
recombinant
techniques. For example, US F'atcnt No. 5,585,089 describes methods for
creating low
iznmunogenicity chiinci.Yc ant.iboclics that retain the high affinity of the
non-human parent
antibody antl contain one or niorc CDRs fzoin a donor iinmunoelol-sulfn and a
framework
regican from a huinan itnmunoglobulin. Unit.od States publication no.
20030039649
describes a humanisation znethod for creating low imntunogcnioity c;himetic
antibodies
containing CDR sequences from a non-human ant.ibody arid fratriework sequences
of
human antibodies tiased on using ca,notticai CDR stt.licturc types of the non-
human
antibody in comparison to germline canonical CDR structure types of human
:tntihodies as
the liasis for selecting the appropriate hunian framework sequences for a
humanised
antibody. Accurdingly, these principles can be applied to the g.rafting of one
or more New
World primate CDRs into a non-New World primate acceptor variable rcgion.
The CDR sequences may be obtained from the genomic. DNA isolated from an
atitibody,
or from seqttelzces prescirt in a clatabasc e.g. Thc Natiunttl Centre for
liiotecluiology
lnformation protein and nucleotidc databases, The Kabat Database of Sequences
of
Proteins of Invmutaological Interest. The CDR scqtience may be a genoniic t]NA
or a
cUNA.
Methods for gra:tt,ing a rcplaccmcnt CDR(s) into an acceptor variable sequence
will bc
faniiliar to persons skilled in tlic art of the present invention.
'I'ypically, the CDRs will be
grafted into acceptor variablc rcgion scquenccs for each Df a vari.able liglrt
chain and a
variable laea.vy cliain or a singlc chain in the case of a dnmain antibody.
The preferred
met.hod of the presetit invcntion involvcs r~,~placemenl of either CDR1 or,
mom prcforably,
CDR2 in a variable rcgion scqucnec via primcr directed mutagenesis. The mcthod
consists
of annealing a synthetic oligonuclcoticlc cncoding S de!:ired mutation to a
target region
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12
whcre it serves as a primer for initiation of DNA synthesis in vitro,
extending thc
oligonuelecytidc by a DNA poly.tnerase to gemerate a double-stranded 13NA that
carries the
desii'ed mutcition, and ligating and clcaning the sequcnce intcr an
appropriate expression
vcctni- (Sambrook, Joseph; and 13avid W. Ru.yseil (2001). Molecular (,'lnning:
A
LrrUoraxvey Manual, 3rd cd., Cold Spriiig lZarhor, N.Y.: Cold Spring Harbor
Labol'atnry
Pi'ess)..
Sti ll Furiltcr, an antibody or anti;en-bi3lding portion thereof may be part
of a larger
immunoadhesic,n molccule, formed bycovalent or noncovalent asscx:iation of the
antibody
or antibody lu-rtiUn with oiie or niore other proteins or Jx;ptides. Examples
of such
immtknoadhesiun tnolecule5 include use of the strepttividin core region to
make a
tctranieric scFv tnolecule (ISipriyancav, S. M., et al. (1995) Human
A.ntlbvdies and
Hyhridamas 6:93-101) and use of a cystciile resitluC, a marker pcptide and zc
C-terminal
polyhistitlizic tag to make bivalent and biotinylrcted sc.Fv molecules
(Kipriyanov, S. M., et
al_ (1994).lvlol. linrrsunol. 31:1047-1058). Antibody poi-tiuns, such a.s Fab
and F(ah')2
1S fragmcnts, can he prepared from wlhole antibodics tzsing conventional
techniques, such as
papain or pepSin dil;cstlon, respectively, oF whole antihocl7es..Moreover,
antibodies,
anlibocly poirtion a and immunuadhesion malecttlcs can tar obta,[:aed using
standard
recombinant DNA t.ccbniques, as described hcrcin as is known to the skilled
arti ,an.
Thc canstant region sequence (Fe portion) is preferably obtained from a human
(ir non-
2() human primate imtiiunnl;lobulin sequence. The primate sequence may be a
New Vtforld
primate or iin Old World primate sequence. Stxitahle Old World primates
.include
chimpanzec, or other horn.inid ape eg. goriTla or orang utan, which because of
their close
pltylogenctic prctximity to huinans; sharc a high degree of homology with the
human
cvnsiant region sequence. Sequences which enc;odc for human or primate
constant regions
25 are rtvailatile from databases including e.g. 't'he National Centrc for
Biotechnology
Information proteirt and nucleotide databa5es, The Kabat Database of
Seclucrtces of
Frott;itis, of Immunological Intei-est.
The antibody or antigcn-binding portion according to the invcttt,ion is
capable of binding to
a humaii or non-humati antigen.
30 Preferably, thc ant.igen tc) which the chimcric antibody or antigen-binding
portion thereof
binds, is peptide, protein, carbohycirate, glycoprotein, lipid or glycolipid
in nature, selected
frotn a tumour-associated antigen including earcinQembryonic antigen, EpCAM,
L.ewis-Y,
l..ewis-Y/I.a, P.MSA, CD20, C.O:10, CD33, C1738, CD52, CD154, EGF-R, I-fer-2,
TR.A11.,
and VEGF receptors, an Antigen involved in an iinnxune or inllammatory
cliscase or
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13
disorder including C:I73, {;'.I74, CD25, C:D40, C.D49d, M1iC. ciass 1, Ml-tC.
class 11, C;M-
CSF, intcrrcron-y, IL-1, IL-12, IL-13, IL-23, TNF-a, and IgE, an antigen
cxpresscd on a
host ccll inclitding glycolarot.ein (.l.b!!lla., P-glycoprotcin, ptirincrgic
recePtors and adhesion
r-eceptt7rs inulu+]inl; CD11a, CD11b, C.DI lc, CD18, CD56, CD5$, CD62 or
CD144, an
antigcn comprising a cytokine, cheniolaine, gowth factor or otiter soluble
physiological
modulator or a receptor thereof includin; ealaxin, I)',6, I'LrB, TGF-P, C3a,
C5a, VF.+GF,
NGF and thcir receptors, an antigen involved in central ncrvous systein
diseases or
disorders includinc (3-amyloid anti prions, an antigen of non-hurnztn origin
such as
microbial, nanobi al or v.i ral a.ntigens or toxins iuclucling respiratory
syncitial viiu5 protein
F, anthrrtx toxin; rattle snakc vcnom and digoxin; wherein thc chimcric
atttibor3y acts as an
agc>nist or antagonist or is active to either deplete (kill or eliminate)
undesired cells (eg_
ariti-CD4) by acting witli complement, or killcr cclls (cg. NK cells) or is
activc as a
cytotoxic agenl or to c:ause Fc-reoeptor binding by a phal;tx:yte or
neutr3lices biological
activity of its target.
More prcfcrably, t.he antigcn is TNPa, prcfcrably huinart TNFm
Altcrnatively the chimeiic antibody or antigcn-binding portion tliereof may
bincl a
non-htaman atatigen. k'referrably the non-human antigen is selected from the
group
consisting of respirat.ory syncytia,l virus F protein, cytomegalov.ix-us,
snatce venonis and
digoxin_
'1'he term "binds to" as used herein, is intended to refer tcr the bindina of
tin antigen by an
immunoglnbulin virriable region of an antibody with a dissociation constant
(Kn) oF 1 Nl
or low~,~r as rncasLtrcc3 by surface plasmon resonance an:.rlysis using, for
example a
BlAcorcT"f surface plasmon resonance systcrn iund BIAcoreTM kinetic cvaluation
software
(cg. vcrsion 2.1). The affinity or dissociation constant (KU) for a spccific
binding
intcraction is prcfcrably about 500 nM to about 50 pM, more prcfcrably about
500 nM or
lower, more prcfcrably about 300 nM or lorvcr and proferably at lcast about
300 nM lc)
about 50 pM, about 200 nM to about 50 pM, and morc preferably at least about
100 nM to
ahout 50 pM, about 75 nM to about 50 plvi, hbout 10 nM tc, about 50 pM.
The antibodics of tho prest,}nt invention an; adv:rntageoug in human therapy
because the
likelihood of induction uf a human anti-antibody response will be reduced.
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14
Rccotnbinatlt antibodies prod.uced according to the invent.iot) that hind a
target at-t.igen can
bc idontificd and iso3ated by screening a ccambinatorial ixr.muncyglobulin
library (eg a phage
display library) to isolate library niemher5 that exhibit the desired bintling
specificity and
functional behavinur. It. wil.l be understood that all approaGhes where
antigen-binding
port,ions or derivaLivcs of antihUdies are uscd, cg Fabs, scFv and V doinains
or drnnain
antibodies, lie withiit the scope of the prc;scnt itivention. 'I'he ph~igc
display technique hits
been descrihed extensively in the art and cxatnples of methods aind
cotnpou.nds for
generating and screctting such libraries and affitti.ity maturing the products
of them can be
found in, for example, Barbats et al. (1991) PNAS 88:7978-7982; Clarlcson et
a1. (1991)
Nalure 352:624:628; Dower et al. PCT. 91/17271, U.S. Patcnt No. 5,427,908,
U.S. Patent
Nu. 5,580,717 and EP 527,839; Fuchs et al. (1991) BioPi'echnology 9:1370-1372;
Garrad
ct al. (1991) B i o/Technology 9:1373:1377; Garrard et al. PCT WO 92/09690;
Gratn et al _
(1992) PNAS 89:3576-3580; Crriffiths et al. (1993) FMBU J 12:725:734;
Griitiths et al.
U.S. Patent No. 5,885,793 and EP 589,877; I=lawkins et al. (1992) J Mol Biol
226:859-896;
I lay et al, (1992) FInm Antihod Hybr.ydanias 3:81-95; (-Ioc>genboom et al.
(1991) Nuo Acid
Res 19:4133-4137; I.luse et Ltil. (1989) Sciencc 246:1275-1281; Knappik et al.
(7000) J Mol
Bitfl 296:57-86; Knappik et al, PCT WO 97/Uti3'70; 'L,adner cL al. U.S. Patent
N.
5,223,409, No. 5,403,484, No. 5,571,698, No. 5,837,500 and EP 436,597;
McCafferty et
ai. (1990) Natzire 34S:552-554; McCafferty et al. E'CT. WO 92101047, U.S.
Patent No.
5,969,108 and EP 589,877; Salfeld et a1. Pc;71' WO 97/29131, U.S. Provisional
Application
No. 60/126,603; a.nd Winter ct al. PCT WO 92/20791 and EP 368,684;
Recombinant libraries expressing the antibodies of thc invention can be
expressed on the
surface of micrcwrganisms eg. yeast or bactcria (see PCT pu.blicaticros
W099/36569 and
98/49286).
The Selected I..ymphocyte Antibody Mctltod or SLAM as it is referrccl to in
the state of the
art, is another means of generating high affinity mntibadies rapidly. Unlikc
phage displrry
apprcaaches all antibodies are fully divalent. In order to generato 1'dew
World primate
antihodics, New World primates arc imtntanlsed with a human antigen eg. a TNFa
polypeptidc. Following imrnunisation cells are removed and 5elcctivcly
proliferated in
individu<<1 micro wells. Supematants arc removed from wells and testcd for
both binding
and function. Gcite sequences can be recovered for subsequent manipulations
eg.
humanisaLion, Fab fragment, scFv or domt3in antibody generation. Thus another
example
is the derivation of the ligand of thc invcntion by SLAM and its derivatives
(BabcoolC, J.S.
et al 1996, Proc. Nati. Acad. Sci, USA 93; 7843-7848, US Patent 5,627,052 attd
PCT
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publication W092/02651). Aclaptations of SLAIvZ, sucla as the use of
alternatives to testing
sulaernat.ants sucli tis panning, also lie within the scope of this invention.
In otte expression system lhe recombinant pept.ide/protein library is
displayed on
rat;tosonies (for= examples see Roberts, RW and Szostak, .1. W.1 997.
5 Proc.l'JatJ.Acad..Se:i.USA. 94:12297 ,- 123202 and P('.'C Publication No.
W098/31700).
Thus another example irivolvcs the generation and iri vitro transcription of a
DNA library
(eg of antibodies and deriVatives) preferably prepared from immunised cells,
but not so
iimited), tr=etnslat.ion of the library such that the protein and ' immunised"
rriRNAs stay on
the ribosum, affitiity selection (eg by binding to RSP), mRNA
isolation,lLVcrsc
10 transltttion and subsequent amplifictttion (cg by polymcrase cllain
reaction or related
technology). Additional rOunds of sclcction and amplifTcation can be coupled
as necessary
to affrnity niaturatie>n lhrough introduction of somatic mutation in this
system or by other
methods of affinity maturation as k-iown in the state of the art.
Another examplC secs thc application of emulsian comparlmentxlisation
technology to the
15 generation of the antibodics of th,e invention_ In emulsion cornptu-
tmentalisation, in viiro
and oplicttl sortiiig mcthods a.re comhiried with e:o-compartmentalisation of
trttnslat.cd
protein and its ntnclcotitle coding sequence in aqueous phase within an oil
droplet in an
tmulsioty (sec PCT put-tlicat.ions na's W099026711 and W00040712). The main
clelnents
for thc gencration and selection of antibudieti are essentialiy similar to the
in vitr n method
of ribosotnc display.
The antibody or antigen-binding portion thereof according to the invention can
be
der.ivati5ed rn= linked tu another functional molecule. For examp,le, the
antibody or antigen-
binding portion can be functionally Iitiked by clae.rnical coupling, genetic
fusion,
ntmcovalent a.ssocihtion or ot,hcrwise, to one or Tnor=e other molecular
entities, such as
another antibody, a detectable agcnt, a cytotoxic agent, a pharmaceutical
sgent, antl/or a
protein orpcptidc that can mediate a5sociation of the antibody or antigon-
binding portion
thereof with anothcr tnolecule (stich as a strePtavidin cnre region or
a,polyhistidinc tag).
Useful cletec;tablc agents with wtllch an antibody or antigen-binding portion
thereof may be
derivalised include fluot'csccnt compounds. Exect)pJary fluorescent detectable
agents
include fiuorescein, fluorescein isothiocyanate, rhociamine, S-dimethylamine-l-
napthalene:;ulfonyl chlcrridc, phycoeiythrin and the like. An antihody may
also be
derivatitaed with detectable cn2ymcs such as alkaline phosphatase, horseradish
peroxidase,
glucose caxidase and the liko. When an antibody is derivatized with a
deteetable enzyme, it
is detected by adding additional teagents that the enzyme uses to produce a
detectuble
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16
reaction product. An antibody ni0y also be dcrivltised with biotin, and
clctcetud t.llrough
indirect measurement of avidin or streptavidin bitltling.
The pre5enl inverttion also extends to *PEGylatcd antibodies or antibody-
binding po.ition
which provide increased half-life and resistance to degradation witltotit a
loss in activity
(eg- binding affinity) relative to non-I'EGy]ated antibody polypeptides.
'1'he antibotiy or aittibody-binding portion as described herein can be
coupled, using
methods knowti in the art, to polymer molecules (preferably PEG) usefixl for
achieving the
increased half-life a.nci degi-iidation resistance properties. Polymer
moieties which can be
utiliscd in the invention can be synthetic or naturally oceuiYting and
include, but are not
liniited to, straight or branchcd chain lxolyalkylcnc, polyalkenylene or
polyoxyalkylene
polyrnes:s,, or a branched or unbranched polysacchaiYde such as a homo-or
heterolxolysacchaiide. Preferred examplcs of syntlietic polymers which can be
used in the
invention includc straiglit or britnche[l chain poly(ethylene glycol) (PEG),
po}y(propylene
glycol), or poly(vinyl alcohol) and derivatives or substituted foriils
t.heteof. Particularly
preferred substituted polymei-s for linkage to antibodies as described her.ein
include
substitttted PEG, including methoxy(polyethylene glyeol). Nat.tirally occu--
ring lx)iymer
moicties which can be used in t3dditiotZ to or in place of PEG include
lactose, amylose,
dextratt, or glycogen, as well tts dcrivatives thereof which would. be
recognised by persons
skilled in the art.
Derivatized forms of polymcr inolecules include, for example, derivatives
which have
additional moieties or reactive groups pi-esent thcrein to Ivi-mit interaction
with amino acid
residues of the antibody polypeptides describcd herein. Such derivatives
includc N-
hydreaxylsuccinimidc (Ni1S) active estcrs, succinimidyl propionate polymcrs,
and
sulfhydryl-sclcctive reactive agenls such as lnaleimide, vinyl sulfonc; and
thiol.
Particularly piefcrred derivatized polymers incl.ude, but are not litnit.cd to
PEG polymers
having the formulae: PI/CJ-O-CHzCHzCH2-COz-NHS; F'EG-O-CH2-Nl 1S; PECt-O-
CH2CH2-C02-N.11,S; PhG-S-CHZCHZ-CO-N.E.IS; PEG-O2CNH-CH(R)-CO2-NF15; PHG-
NHC:O-CHZCH2-CC7-NHS; and PEG-O-Cf12-C(72-NEIS; where R is
(C,H2)n)NHCO2(rnl.''EG)= P1;G polymers ean be linear molec:ules, or can be
branched
whet-ein multiplc PEG moieties are present in a single polymer.
The reactive group (e.g., MAL, NHS, SPA, VS, or'l'hiol) may be attached
directly to the
PF3Ci polymer or may be attached to PEG via a linker molecule.
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17
The size of polynlel's useful in the invention can be in the rangc of between
500I1a to 60
kt]a, for exatnple, between 1000 Da and 60 kDa, 10 kDa and 60 kDa, 20 kUa and
60 kDa,
30 kDa and 60 kDa, 40 kDa and 60 kDa, ancl up to between 50 kDa and 60 kDa.
The
pOlymers uscd in the invention, particularly PFCr, can be straight chain
polymers or may
possess a branched conformation.
The polymer (PEG) mOleeulcs uscful in the invention can be attuched to an
antibody or
antigen-binding portion thercof usitig method5 which arc well known in the
art. The first
step in the attachment of PEG or other polymer moieties to an antibociy
polypeptide ,
fnononier oi-multimcr of the invention is the substit.ution of the hydroxyl
cnd-groups of the
P.F..C'r polymer by clectrophile-containing functional groups. Particularly,
PRCir polymers
are attached to cither cysteine or lysine residues present in the antibody
Polypeptide
monomers or multimers. The cystci:nc and lysine residues can be naturally
occurring, or
can bc engineered into the antibody polypeptide molectile. For example,
cysteinc residues
can be recomhinuntly englneercd at the c:-terminus of an antibody polypeptidc,
or residues
at specific solvent accessible locations in an antibody polypeptide can be
substituted with
cysteine or lysine.
The anti body may be linkcd to one or more molccttlcs which can increase its
half-life irt
vivo. "['hese molecules arc linked to the antibody at a site on the antibody
other than the
antigen binding site, so that they do not inlerfcre/sterically hinder tht
9ntigen-binding site.
Typically, such tnolccules are pelypeptides which occur naturally in vivo and
which resist
degradation or rcnloval by endogenaus mechanisnas, tt wi I I be obvious to one
ski l led in lhe
a,t that fragmcnts or derivatives of such naturally occumng molecules may be
used, and
that some may not he polypeptides. Molecules which increase half life may be
selecled
from the folI owing:
(a) proteins frorn the Cxtracellular mrttiix, eg. collagen, iaminin,
intel,*rin and
fibronecti n;
(b) proteins found in blood, eg. fibrin a-2 rnacroglobu3in, serum albumin,
fibrinogen A, fibrinogcn B, serum amyloid protein A, hcptaglobin, protein,
ubiquitin,
uteroglobulin, 8-2 microglobulin, plasminogen, lysoLymc, cystatin C, alpha-l-
antitrypsin
and pancreatic kypsin inhibitor;
(c) imtnune serum proteitls, cg. 1g.F., IgC7, igM;
(d) transport proteins, cg. rctinol binding protein, a-1 microg.lobulin;
(e) defensins, eg. bcta-dcfensin .l,lVeutrophil defcnsins 1, 2 and 3;
(f) proteins found at the blood bt'ain barrier or in neural tissues, eg-
melanocortin
rcccpt.or, myelin, ascorbatc tt=ansporter;
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is
(g) transferrin rt;cept.o.t speci fic ligand-Deurol,harmaccutical agent
ftision prnteins
(see 1,JS5977307); brain capillary endothclial cell receptor, transferrin,
transferrin rec;tptor,
it3sulin, insulin- Jike growth factor 7(IGP 1) receptor, inSuli21-like growth
fnetor 2(IGF 2)
receptor, insulin receptor;
(h) proteins localised to the kidney, eg- polycystin, type IV collagen,
organic anion
transportcr K1, 1-leymatul's antigen;
(i) proteins localise(I to the liver, eg, alcohol clchydrogenase, G250;
(j) blood coagulation fktctcrr X;
(k) rx-1 antitrypsin;
1() (1) HNF 1cx;
(m) proteins Icx:aliscd to the lung, eg. secretory component (hinds IgA);
(n) proteins lcx;i~discd to the Heart,cg. IISF 27;
(o) proteins localised to the skin, eg, ker-atin;
(p) hone apecific proteins, such as 1?pne morphogenic proteins (BMPs) eg. SMP-
=22,
-4, -5, -6, -7 (also rcfcrred to as ostcogeDic protein (OP-1) v.nd -S (OF-2);
(q) tumour specific pn7teins, eg, human trophoblast antigen, herceptin
receptor,
oestroge.n receptor, cathepsinr; cb cathep5in 13 (found in liver and spleen);
(r) disease-specific protcins, eg, antigens cxpressed only on activated T-
cclls;
including LAG-3 (lymPhocyte activation gene); osteoprotegerin ligand (OPGL)
see Nature
402, 304-309, 1999; O.]C40 (a tncmber of the TNFa receptor fanlily, expressed
on activated
T cells and the only costimulatory T cell molccalo known to be specifically up-
rcgulated in
human '[' cell leukaemia virris type-I (H"fLV-I)-producing cells - see J_
Irnmunol. 2000 Jul
1;I6561):263-70; melalloprotea=ses (associated with aithritis/eancers),
including CC.'r6512
.Drosophila, human paraplegin, human FtsH, huanan AFG3L2, murine flsH;
angiog,enie:
gi-owth factors, including acidic fibroblast growth factor (FGF-j), basic
fibroblast growth
factor (P"G.F-2), Vascular endotheliat growth factcar/vascular perrneability
factor
(VEGP/VPF+), transfortning growth factor-cx ('f'GF-oc), tumor necrosis factor-
alpha ('INFa),
angiogenin, interlcukin-3 (tL-3), intcrleukin-g (TL-8), platelet derived
endothelial growth
factor (l?.D- SCGF), placental growth factor (PIGF), tnidkine platelet-derived
growth
factor-f3li (PDGF), fractttlkine;
(s) stress proteins (heat shock proteins);
(t) pr=oteins involved in Fc transport; and
(u) vitamins eg B12, 13iotin.
fn another aspect, the invetltion provides a pharmaceutical composition
Comprisinl; an
effectivc amount of the chi.meric antibody or antigen-binding portion thcreof
according to
the present invention, together with a pltarniaceutically acceptable excipient
or diluent.
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19
A"phtirniacetltically acceptable excipient or cliluent" itlcludes any and ti11
solvents,
dispersion media, coatings, aniibacterial and antifungal agents, isotonic and
ahsolption
dcluying agents, and the like that are physioiogioally Gotnpatiblc.
Exa.r.npics t>f
pltarmacuuticalty acLeptable carriers include one oi-morc: of wat.et, salinc,
phosplizte
buffei-ed saline, dextrcrse, glycerol, et.hanol, anc3 the like as well as
combinaticro% thereof.
In many cases it will be pre.ferable to include isotonic agents, for example,
sugars,
polyatcohols such. as rnannitol, sorhitol, or sodiiirn chloridc in t1le
composition.
Phtarmaceutically acceptable substances stich as wetting or tninor 3triounts
of a.uxiliary
substances such as wetting or e.tnulsi fyi rtg a;ents, preservatives or,
btiffers.
7'he term "eftect'ive amount" refers to }ln '=ltnollnt of Atl antibody i)r
antigen binding lxmt;tion
thereof (including phayTmaceutical compo5iti.ons cxoinprising the antihody or
antigc)
bincling porticm tfiercof) sutficicnt to trcaat a specified disettse or
disurde.r or une or tnore ot
it.s symptoms and/or to prevent the caccurrc;nce of the disease or disorder.
Tktc torm "diagnostically efFective amount" or "wnounts effcetive for
diagnosis" and
cognates thereof, refers to an aniount cif a antibody or untigen binding
pcNrtian thereof
(including ph4trrnaceutical compositicn,s con-,prisi.ng the antibody or
antigen binding
portion ihereof) sufficient to c{iagnose a spccified cliscase or disorder
and/or oiye or inore oF
its Tllallifestatlons, where diagnosis includes identilication of the
existence of the diseaso or
disordcr anc!/or tlotec:tiott of the extcnt or severity of thc; disease or
disorder. Often,
diagnosis wi11 lv Carried out with refGrence to a baseline or background
detection level
observed for individualS without the disease or disorder. Levels oFdet.ection
above
background or bascline levels (elevated levels of detection) are, indicative
of the presence
ancl, in some cases, thc severity Crf the condition.
When used with respect to inethods of treatment and the use of the antibody or
antigen
biliding portaon thereof (including pharrnaceutical comlaositions comprising
the antibody
or antigen binding poil,ion thereof), an individual "in need thereofi' may be
an individual
who has been clial, iosed with or pY'eviou.sly treated for the disease or
disordcr to be t.ecttted.
With respect to methods of diagnosis, an individual "in need thereof"may be an
individual
who is suspectcd to havc a disease or disorder, is at risk for a disease or
disorder, or has
previously been diagnosed with the disease or disorder (e.g., dia.ginosis can
includee
tnottitoring of the sevcYf ty (e.g., progre.ssion/regression) of the disease
or disorder over
tijxxt and/or in conjunction with ther;.ipy).
It is preferred that the ehimeric antibody or antigen-binding pc}rtian thereof
blocks or
stimulates rcecptors functions or tx;utraiizcs activc soluble products, such
as one or more
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of tFze interleulcins, TNFa or C5a. M.oi'a preferat)ly, the a.ctive soluble
product is human
'I'N Fa.
The composition may he in a vc,riety of forms, including liquid, scmi-solicl
or solid dosage
forms, such as liquid solutions (eg injectablc tind irt}ttsible solutions),
dispcrsicans or
5 suspensi071s, tablets, pilts, poWdci=S, lipqsonics or suppositories.
Preferably, the
composition is in the fonT- of an injectable solution for immunAzation. 'f'hc
aclniinistration
inay be intravenous, subcutaneous, intraperitoneail, intrainuscular,
transdennal, intrathecal,
and intra,artcrial. Preferably the dosage fortn is in the range of }rom about
0.001 mg to
about 10 m_g/1Ãg body weight administered daily, weekly, bi- oi- tri-weekly or
nicinthly,
10 moc'c preferably about 0.05 to about 5 mg/kg body weight weckly.
The compusi tioti niay also be formulated as ct sterile powder for the
prepiratinn of steriie
.ir5jectablc solutions.
In oertain einbodiments, the active compound may be prepared with a carricr
that will
protect the cotnpound against rapid retea.5e, such as a controlled release
formtrla.tion,
15 including implants, traaisclermal patches, and tnicroencapsulated delivery
systems.
t;ompiitibie polymers may be u.sed such w; ettiylcne vinyl acetate,
polyanhydrides,
polyblycolic acid, collagen, polyortrioosters or pulylactic acid.
'I'fte composition may a.lso be form.ulatcd for oral administration. In this
embodiment, the
antibody may bo enclosed in a hard or soft shell gelatin capsule, compressed
into tablets, or
20 incorpot:ated directly intn the subject's diet.
The ccrmposition may also be formulated for rectal administt'ation.
The antibody may be administered in artier to bind to and identify selected
cells iyd vitr
and in vivo, to bind to and destroy selected, cells in vivv, or in orclcr Lo
pettctrate into and
destroy selected cells ira vivo. Alternatively, the antibody may be used as an
immunotoxin
to deliver a cytotoxic agent eg_ a toxin oi- cliemotherapeutic agent to a
pat'ticular cell type
such as a tumotir cell. Production of immunotoxins would be fatnilirrr to
persons skilled in
the art.
Cytotoxic agents cotinmonly used to generate immunotoxins include radioactive
isotopes
such as 1 ".[n or90 Y, sclCnium, libonucleaScs, binditlg tlomain - deleted
truncated micrcfhial
toxins such as Pseudomon<<s exotoxin or I}iphlheria toxin, tubulin inhibitors
suci7 as
calicheamicin (ocagamicin), maytansinoids (incluc.tatlg DM-1), auristatins,
and taxoids,
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21
r,.ibosozne inactivating proteins such a.s ricin, ebulin I, saporin and
gclonin, and prodrugs
such as nlelphalati.
In the preferred ecnborlinient, thc composition is administered to a hturiart.
The present itivent.iotl also pro'videts for the use of the chirneric antibody
or antigen-binding
portion thereof in a diagnostio applie;ation for detecting an antigen
associated with a
particular disease or disordcr.
More particularly, the invention provides for the use of the chimeric antibody
or
ant,igen-bincling portion thereof in a inethocl for diagnosing a suhject
having an antigcn
associatcd w'rtli a particutar disease or disorder, comprising adaninistc;ring
to said subject a
If) diagnostically effective amount of a pharmaGeut.ical composition according
to the third
aspect. Preferably the stlbjcct is ai human.
For example, the chirxieric antibody or antigen-binding fragment thereof,
prcforably
labelled, can he used to clet.ect the prescnce of an antigen, or elevated
levels of an antigen
(e.g. TNFcx) in a hiological. sa.tnplc, such as scrum or plasma using a
eorivent=ion
.15 imtnunoassay, tiucii aLs an enzylue linkcd itnrnUnosorbenL assay (EU'a'A),
a
radioirnmunoar,sa.y (RIA) or tissue itumunohistcx;hemistry.
Preferably, the atitigen to which the chimeric antibody or .antigen-binding
portiUn ther-eof
binds, is pcptrdc, protein, carhohyd.rate, glycoprotein, lipid or glycolipid
in nature, sclectecl
from a tuniour-associatcd antigen includinl; ca-cinoenibryCrniC atltigcn,
EpCAM, Lewis-Y,
20 I,ewis-Y/b, PMSA, CD20, CD30, CD33, CD38, CD52, CD154, EGF-R, Her-2, TRAIL
and VEGP i-eceptors, an antigcn involved in an immune or inflanunatory disease
or
disorder including CD3, CD4, CC17.5, [;D40, CD49d, MHC class I, MHC class t!,
GM-
CSF, interferon--y, I.la-1, IL-12, IL-13., IL-23, TNV-a, and IgF, an antigcn
expressed on a
host cell including glycoprcitein llh/l Ila, P-glyeoprotein, purinergic
receptors and adhesion
25 receptors including CDIla, CD11b, CI)1Ic, CD1.8, CD56, CD58, CD62orCD144,
an
antigen comprising a cytokine, cheutokitic, growth factor or other soluble
physiological
modulator or a receptor thc;reof includ9ng eotaxin, IL-6, IL-8, TCF-J3, C3a,
C5a, VfiC;F,
NGF and their receptors, an antigcn involved in central nervous system
diseases or
disorders including j3-amyloid and prions, an antigen of non-human origin such
as
30 rnicrc,bial, nanobial or viral antigens or toxins inclueling respiratory
syncitial virus protein
P. anthrax toxin, rattle snake venom ancl digoxin; wherein the chimeric
atttibody acts as an
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22
agonist or antagonist or is active to eitlter deplete (kill or climii7tttc)
undesired cells (cg.
;tnti-C134) liy acting with ccmplenicnt, or kiliercells (eg. N'K cells) ot=is
active as a
cyt.ot.oxic a.gent or to cause Fe-receptor bindin~ by a pla3gocytc or
neutralizes biological
ttctivity of its target.
'I'he anti-hunian TNFa chimeric antibody or antigen binding poifiiott thereof
accordtitig to
the inventioti mtty also he used in cul] cultur=e applications wherc it is
dcsired to inliibit
TNFsY activit.y.
The present invetrt.ion also provides a mcthod for treating zt disease or
disordcr
cliaracterised by human TNFa activity in a human subject, comprising
adrxtinistcring to the
subject in need thereof a pharmaceutical cotttposilion according to the
present itivcntion in
which the clvirrteric antihody or atrtigen-hinding portion thcreof bincls
TNlcrx.
The term "diseasc or disorder chat'actcrised by hunlan TNlya ttctivity" as
used herein is
intended to includc diseases or disordcrs in which the presence ofTNFu in a
subject
suffeting frona the discttse or disorder has been showtt to be or is suspected
of being eithcr
responsible for the pathophysiology of tho disease or disorcler or a factor
that contributes to
the worsening of the discase or disorder. Accordingly, a disease or disorclcr
in which
TNFa activity is detriincntal is a disease or disorder in which inhibition of
TNFa activity is
cxpceted to ttl leviate syniptoms and/or progression of the disease or
disorder. Such
diseases or ditiorders may be evidenced, for example, by an increase in the
cortcentration of
TNFa in abiolagicttl fluid af a Subject staffcring frcrni the discase or
disorder (e.g., an
inc.reasc in the ooncentratiotl of TNFa in serum, plasma, synovitt] fluid etc
of the subject),
which can be clctected, for example, using a chimeric antibody of the
invention spccific frn=
7'Nl~a_
A disease or disor.'clcr charttcteri.sed by human TN.Pa ttctivity is intended
to include diseases
or disorders arid othcr disease or disorder in which the presence of TNFa in a
subject
sufFering froni the disease or disorder has been shown to be, or is suspected
of being, either
resgcrosibie for the pathophysiology of the disease or disorder or a factor
which, contributes
to a worsening of the disease or disorder. 'Preferably, the disease or
disorder characterised
by human TNra activity is selected from the group consisting of scpsis,
including septic
shock, enclotoxic shock, grain negative sepsis and toxic shock syndrome;
autoimtnune -
disease, including rheumatoid arthritis, rheutxtfltoid spondylitis,
ostcoarthritis, psoriasis and
gouty arkli-ritis, allergy, multiple sclerosis, autoin'utuine diabetes,
autoimmune uvoitis ttnd
nephrotic syndrome; infectious discase, including fevc;r and rnyalgias due to
infcction and
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23
cachcxia sccondary to infection; graft vctstas host disease; turnour growth or
metastasis;
pulmotlary disease including adillt respiratory distress syndt-ome, shock
lung, chronic
ptllmClnai'y inflammatory disease, pttlnionary sarcoidosis, pulmonary fibrosis
and silicosis;
inflammatory bowel diseaso inclttclittg Crohn's disease and ulcerative
colitis; cardiac
disease; inflammatnry bonc disc3sc, bepatitis, cc,agulation disturblinccs,
bunts, repertu;;ion
injury, keloid formation and scar t.issue formation.
Supplementary aetivt: compot7nds can alsn he incorporated into thc
conlposition, '1'he
antibody or antibody-binding fragment may he co-formulatcd with ancl/or
administered
simultanetausly, scpai-ately or sequentially with one or more addit.ional
therapeutic agents
eg. antibodics that bind to other targGts such as cytokines o.t' cel i surface
molecules or
alt.crnativcly one or mc3t-e chemical agents thZt= inhihit hunian '1'NF(x
prpducstion or activity.
In another aspect, the invont.ion provides a kit compt-ising a therapeutically
effective
amount of a chimeric antibocly or antigen-binding portion of thc invention, or
a
phan-naceutical composition comprising a therapeutically cffcctivc amount of a
chirneric
antibody or antigen-binding portion thereof, togetht r with packaging aitid
instructions for
use. In t;atain c.tnbodint.exits, the instructions fttr usc inclttde
insttvct.ions for how tt)
effectivaly -idmitiister a tt-eraPeutic amount of the chimcrie antibody or
antigen-binding
portion of the invcntion.
Throughottt this specification the word "comprisc", or variations such as
"comprises" or
"comprising", wilI he understood to imply the irlclusion of a stated element,
integer or step,
or grotrp of eletnents, integers or stcps, but not the exclusion of any other
element, intcgcr
or step, or group of elemcnts, integers or steps.
All publicittiuns incntioncd in this specification are herein incorporatccl by
reference. Any
discussion of documents, acts, ti}aterials, d.evices, arlicles or the; likc
whiclt has been
included in the prescnt specification is solely for the purpose of providing a
co,ntext for the
present invcntion. IL is not to be taken as an aclmission that any or all of
these mattert; form
part of the prior art base or wet=e common general knowlec3gc in the field
lelevant to the
present invcntion as it cxisted in Australia or elsewhere before thc priority
dat.e of each
claim of this application.
In c.-rder lhat the naturc of the present invention may be more clearly
undcrstood, preferred
forms thereof will now be described with reference to the follawing non-
limiting
examples.
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'74
i'+'.Xr1MPLI';1
Fusion ora marmoset variable region to a huatiati constant region
Materials aiici niethixL;
C;ene Synthe.si.c aratl Cloning
The VH c.hain (Accession Numl:jer: t1AM54057, SEQ LD NO: t) of the MOG
specit;tc
tnarmo5et defived antil?ody wa,; expressc;cl with 3 human constant region
(hutnan Igt.Y I.
heavy chain Citl, hinge, C112 &. CH;i domains (such as 'NGBI accession numl7er
P01$57)
(SEQ ID NCa: 2)). This was achieved by back translatiun of the amirro acid
sequence into a
DNA sequence which was optimized for tnatnmalian cell expression using
GeneUptimizer
tc:chnology and synt.tiesized de rtavv by aSsentbly of synthetic
oligonucleotidCs (CleneArt,
Germany). During DNA sequence optimisat,ion the specific restriction enzyme
sites Asc I
atld 2kh 1.111 were included to allow for future manipulation of the VH
regiotl. Foilowing
gene synthesis the whole sequence inclucfing a Kozak secluc,~nce was eluned
into the
multiple cloning 5ite of the pEE6.4 GS accessoiy vector (Lonca. I.3iologics).
Thc V,, chain
(Acces:aicm Nutnber: AAM54058, SEQ ID NO: 3) of the MOO specific marmoset
derived
antibody was expressed with a hunxau kappa iiglit chain constxnt region (such
as NC[3[
accession nuinbcr AAA58989) (SEQ ID NO: 4). DNA encoding the light chAin (VL-
Kappa) amino acid seclueace wa4 prepared as described above for tlic hcavy
chain. During
/
DNA sequence optimii..ation and synthesis the specific restriction enzytne
sites Bsi Wi
Rsr 11 were included to allow fttture manipulation of the VL region. Following
genc
synthesis the whole setluence inGluding a Kozak sequcnce wa.s cloned into the
multiple
cloning site of the pEE12.4 GS exprc;5sion vector (Lonza Biologics). For
stable expression
thc two singlc gene vectors (pEiF.6.4-VH-IgGt and pFir12.4-VT,,Kappa) werc
combined into
a double genc vec:tor. This wati done by digesting out ol'the pEE6.4 backbonc
the heavy
chain expression cassette (hCM V-MIE promotcr, Kozak sequence, ma.rmoset VH,
human
constant region and SV40 polyA sitc) using No! I andRrr.niH 1_ The resuitant
fral;ment was
subotoned using Not I and Bcarnkl I sites into the pE'F..12,4-Vr, Xappa vector
downstream oF
the light cilain expression cassette (hCMV-MIE pminoter, Kozak scquence,
marmosct Vtõ
humitn Kappa constaiit re&ion and 5V40 polyA site) creating a vector
expressing both the
hoavy and light chains of AB 138 (SEQ II] NOs: 5 aud 6).
Ti-u.rz.sfecX don
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For each t.ransfectiun 175ft1 of Lipofectaminc 200() was acided to 5trzi, of
Optirnetn I inedia
(Invitrogen Cat Nos. 11668-027 and 31985-062) in a well of a 6 weli plate. In
a secoiul
well 70 I of the expre:ssioit vector (70 g) was added to 5 rrtL of Optimem I
media.
Following a$ niinutc room ternperature incubation, the contents of the two wt
1ls were
5 mixed together ancl lcft for a futther 20 rnitiute inGuhation. Following
thir; seconcl
incubation the whole transfeGtion mixttirc was added to a T175 tissue culture
flask
containing the CHOKISV cel15. Cells were inctihatet{ for 72 to 90 hours tiricl
sttpornatants
harvested. Supt;m3t.ants were centriftrgcd at 4,000 x g for 5 tltinutes to
pcllct ccll debris,
a.ncl were filter sterilised through 0.22 p.m caiti-idge filter.
10 Antibucly Purif'icczti.nra
The supernatant was passed over a HiTrap Protein A colcnnn (Amersh:rm
Biosciences, Cat.
No: 17-(1402-01) thrce times at a flow tate of 1 mUmin. The column was then
washed
with 20 niM strdium phosphate for 40 s2tins at I m=Umin. The antibody was
cluted with 0.)
M citric acid pH 3.5 with fractions collected and immediately neuti-alisecl
with 1M Tris-
15 Ilt;'.l pl-T 9Ø Antibody samples were then desa.ltecl on a PD-I O column
(Amcrsham
13iosciences, Cat No: 17-0851-01). Analysis of the anribocly by SUS-PAGE and
size-
exclu5ion TULC confirmed the coYY=cct molecular wcight, presence of assembled
antilxady
and the concentration of antiboil=y.
Westenr. Blot analysis
20 The ability of AB l38 to retain binding to the antigeD of M26, rat MOG
(myelin-
Uligodendrocyte glycoprotcin), was investigated by Westem Blot. 130 mg of rat
spinal
cord (IMVS, Australia) was hnmogenizcd in 1.8 ml CelLytic M Cell l.ysis
lZeagent
(S1GMA, C2978) and iiicubated for 30 minutes at 4 C. Further homogenization
was
pei:t'crnned by drawing the lysate through a 27g112 net;dlc scveral times
followed by
25 ceaitrifugation at 4"C and 13000g for 30 minutes. The pellet and
supernatant was cliluted
into SDS-PAGE sample buffer (125 mM Tris-HCI pH 6.8, 5~1a SDS, 0.25~'io
bromophenol
blue, 25'~'o glycerol). Along with this 200 p.l CHOKISV cells at I X 106
viable cells per nil
were ,spun down at.1300t) x g at 4 C for I tninute and resuspettded in 200 gl
CelLytic M
Cell Lysis Reagent (SIGMA). Following centrifugation at 4 C and 13000 x g for
30
minutes the superziat9nt wa.s mixed with the apprcpri:rtr; amouiit of Sl7S-
PAGE sample
buffer. All samples, along with a sarnple of xnolecular weight markers, were
run on a 4-
20%1 Novex pre-cast gel (]nvitrcrgen, Australia) for 2 hours at 120V. Proteins
were thcn
transferi-ed to PVDF (13ioRad, Australia) using a western blot apparatus in 1
X Tris-
Glycine l3uffer with 20e~'o methanol (BioRad, Cat 161t-0771) at 4 C- at 250 mA
for2
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26
hours. '1'he me.mbrano was then b.locketl by inculiat.iott with 5 c;"a skim
milk powder in PBS
for 1 h at rnoni tetnpcz'ature, 'I'he rnctnbrane was then washed with t X PBS
three tinles
followed by an overnight incubation at 4"C: with AB 138 in PBS at lU uglmI...
After
washing, the membrane wa5 incubated with Goat Anti-human I'gCr (I.i.+L) HRP
cnnjttgate
(Sigma, Australia) dilutcd 1:5000 in I XPBS for t hour at ronm temperaturc.
Fol#owing
washing, bocmd a3itibody was detected using the FCL Wcstcrn $lntting AnalySis
Systenl,
(Amerstiam Biosciences Cat: RPN21 09). A parallel eApcritncnt waL,;
pexforxticd in which
A13138 was replaced witlt an isolype-matchcd irrelevant specilicity negative
contrnl
antibody (anti-TNNnc tnonoclontil antibody) in order to identify any non-
specific binding
events.
Results
After suocessfu) protein cxpressic>n and purification, westcrn blot analysis
was performed
on AB138 to Lleternnine if it rctitined binding affinity to rat MOG. AB138
bound a protein
with approxitnitte size of 25 kDa present in the rtit sPinal co.rd cleared
lysatc, a protein not
Prese.nt in clearcd CI-IOKISV lysttte (higtirc 1). The negative control
anti.body did not .h.i.nd
to protein present in either Iysate jndicating t.hc interaction betwcen AE313H
and the protein
of size 25 kDa was not due to artifact or non-spe,uific binding events
associated with the
huma.n constant regicm (Figure 2). This protein matches the expccted size of
rat MfJG
minus the signal setluence (24.9 1cD~k).'1'his result indicates that AB138
retttincd affinity
for rat MC)C, present in rat sPinal cord lysate and demnnsti-ates that a
marmoset human
fusion antibody can retLtin antigen bir,ding al~ility.
It can bc tippreciated by somecme skilled in the act that rat MOG could bo
produced using
recombinant DNA teclinology and the ability of AT3138 to bind rat MQG
determined it)
binding assays such as ELISA or 13iacore analysis.
EXAMPLE 2
CDR2 Substitution of a domain antibody
Standarcl reeornbinant DNA technoJogy can he used to procluce a locally
engincered
dor.uain antibody by substitution of the CDR2 of an acceptor anti-TNra domain
antibody
(13asr,tn et al. WO 20()4/081026; SFQ Ip NC-: 7; Figure 3) with a CDR2 from a
donor Naw
World primatc immunoglobulin.
Applying thc rules of Kabat (Sequences o!'T'roteins of Itnmunological
Intcrest" k:. Kabat et
al_, U.S. Dcpartment of Health and Human Services, 1983) the CDR2 is
idc;ntified on the
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27
acceptor anti-TNF-a dnmain antibody (SASFLQS). The domain antibody acceptor
scquence is then aligned against a panel of lVew World primate immunogloUulin
secluences. These sequences are derived fi-om the Ma's tiight. tronkey (Aottis
nancymaac)
(SFQ fD NOs:, 8 - 113) and from the common marmosct (Cal.lithrix,jacchus)
(S.FQ lD NOs:
la - 24 ) (Figurc 4). '1'ht CDR2 sequences of the New World primate
ilrununoglnbulins
ttia.t differ from that of thc acceptor C:DCt2 5equcncc can lse identified as
SASTLQT,
,DASSr.QP, GASTRAT, KVSNRAS, KVSNRA.S, KVSTRGP, AASNRAS,'I :SSNLQA,
KASTLQS, AAS'I'L,QS, YASSLQS, YASFLQG) (Table 1). I3LAST analysis
(http://www.aichi.nlrrt.uih.govlBLAST/) oYt each of these donor New World
primatc CI7R2
I0 sequcnces is Perfcartncd to remove sequences that are exact matches for
human
itruntx.noglnbulin sequences. Setlucnces unique tca New World primates wure
KVSNRAS,
RVSNRAS, KVSTR[,7P, AASNRAS, TSSNLQA, DASSLQP, YASFLQG (Table 1)_
SL:Q CDR2 Contp:~rison 19X~AST analysis
il.? NO sequence t4 acceptor against I.Iomo sapien
sequencc sequences
~iEL(iFS)
(SASELQS)
!1 KVSNRAS pifferent No ex,~ct matchcti
9 KASI'i.QS Differer~t Matches huutnti
AAS'1'LQS Diffcrent Matches hurn:in
11 AASNRAS Diffcrcnt No exact rnatches
12 ''SSNT.Qi1 l~ifferant Na cxfict matches
13 YASSC,QS ED=
Matchas humtui
14 YASF~L(~C~ Dificrent No exact rnatehes
RVSNRAS 1)ifferent No Cuct matches
16 KAS"CT.QS Different Matches hum an
17 t;AS'J'khT Diftereot Matchcs human
1$ KVSTRGP Difforcnt No exact matches
19 SASTLQT Different Mntcltes human
CTA.r.,TRAT 1)ifferent Matches }lpman
21 DASSLQP Diflerent No exact matchcs
2'2 GASTRAT Diffcrent Matrhes human
23 GASTRAT f)ifferent Matches human
24 CiAS'MAT Dil'fcrent Matchcs hun~n
Table 1: New World primate CDR2 sequences and their suitability as dcmor
sequcnces.
15 The acceptor CDR2 and the potential donor CDR2s dre exatnined for their
predicted
imtnunogenicity in hulnans by the MHC class l[ binding prediction progrum
Propred
(http:/Iwww.imtech:res.in/ra h~ uv,,~/nroprc(l) using a 1% threshold valtie
analysis of all
allclcs. From this analysis the acceptor CDR2, SASSLQS, forms part of the
peptide,
LIYSAS[:LQ, which is predicted to bind Ml1.C class II encoded by 1 I alleles
20 (DRBl,_0306, DRB1_0:307, t7RB1_0308, DR131_0311, DRB1_,0401, DIrB1_0426,
CA 02619245 2008-02-12
WO 2007/019621 PCT/AU2006/001166
28
t]RB1_I)806, DRIi 1_0813, nRB 1_i 501, DRt31,._1502, DRB 1_ 1,506). 'f'he
donor CDR2
5equcnce, KVSI'JRAS, .forms part of a seqiu;nce, LIYKVSNRAS, which is
pi'cclicted to
hind MHC; class 11 encoded by 9 alleleS (DRb1_(.)309, DRB l_O4f.)2, DRi3l
0802,
URFi 1_0804, DRB 1..9806, URBI 0813, I?RF31_1301, DRE l_1327, DRt3 t_1328),
Ttic
donor CDR2 scquenee, AASNRAS, forms part ot a sequencc, LYYAASNR.A, which is
predictcd to bind Mt-.iC class ll encoded by 6 alleles (D]ti3l_0402,
I7RF31_0404,
D3tt31_0409, I7RB1_0423, DRB1_0813, D,RB1_1506).'!'he donorCDR2 seqttance,
TSSNLQA, forms pait of a sequencc, LIYTSSNLQA, which is predictid to bind Mt-
IC
class 1[ encoded by 10 alleles (llR131_0401, DRB 1_0402, URB1_0404,
DItB.1,_0410,
.DRB]_0423,'llR131_0426, DRB1_()813, DR131_1501., DR131_1502, DRB1_1506). The
donor C:DR2 sequence, KVSTRGP, forms purt of a sequence 'Lt.IYKVSTR, which is
predicted to i.iind MXIC cla:,s II encoded by 8 alleleS (UR131 03()9,
DR131,0802,
D'R131 0804, DR$1 ()80fi, DR131_0813, X7RB 1_1.301,17R131_1127, DRB 1_1328).1-
Icnce,
the acccptur CDR2 can be rc platced with a donor C:i.7R2 nf lowcr predicted
irnmunogcnicity, including KVSNRAS, AASNRAS, TSSNI.,QA and KVS t'RGP.
Using recombinant DNA t.ec}anolc>gy, the acceptor C::OR2 is replaced with the
donor CDR2
sequences, generating the locally enginctred domain antibadies (SEQ ID No: 25 -
31).
Examples of recombinunt DNA technology include k)tosc ciescribod by Winter et
aI. (t,1S
5,225,539), and include, but is not iiniitod to, techniques such as site-
ci,irected mutagenesis
and oligo annealing. Protein exprewsion of the d4main antibodies is then
perfnnncd in =F.
_coli BL21(DE3) pLys (Novagen, Germany) using.a su.it.a.ble vector for
expression such as
pET21d(+) (Novagen, Germasiy), or by ot.her such matjiods known in the art
such as thnse
describc by 13asran et ctl. (WO 2004/091026). Nollowing liacteriml ccll lysis
the domain
antibodies are purified using Pi-otein L(Piercc:, 13SA) chro.tn3tol;raphy.
Following purification the enbineered clomain antiboclies arc analysed for
retention of
TNFa binding ability by incthods known in the art, such as the L929 ucutral
isatiun assay
or the'f'NFrz receptor I hindin.g assay.
To improvc the binding affinity of thc engineered dottiain axltitwdies,
affinity maturation
could be pcrforrnccl by amino i1cid silbstitutiotl of the frunieWork residUes
SUTL'OUnding and
stttbilising CT?R2 or by othcr methods known in the 3r't.('Wintcr et al. (US
5,225,539);
Griffiths et cal (US 5,885,793); ltajpal, A_ et cil. (2005) Agencral method
for greatly
improving the ai"tinity of antibadies by using comb.inatoria.l Iibraries, Proc
Nail Acad Sci U
S A., 102(24) 8466-71; irving R.A. et ccl. (2001)12ibosome display and
affinity maturation:
from antibodies to single V-doniairxs and steps towards catlcCr thei~apeulics,
Jaurnal of
Immunological Methods, 248: 31-45).
CA 02619245 2008-02-12
WO 2007/019621 PCT/AU2006/001166
29
ft wi{I be appreciated by I,ersons skillcd in tht: art lhat nume.t'ous
variations and/or
mc>dificatioits may be made to the inveiitian as showii in the specifio
r;mbndim,ezits withoul
departing from the spirit or scope of the invcntivn as broadly cleseribeci.
't'he present
embodiments arc, thc;refare, to be considered in all respects as illttstrative
and npt
restrictive,
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